ucb cell lines  (ATCC)

Journal: International Journal of Molecular Sciences

Article Title: The Role of Interleukin-1-Receptor-Antagonist in Bladder Cancer Cell Migration and Invasion

doi: 10.3390/ijms22115875

Figure Lengend Snippet: IL1RA expression is absent in invasive bladder cancer cell lines. ( A ) IL1RN mRNA levels in freshly isolated benign patient ureteral urothelium (#1, #2, #3 indicate individual sample numbers), and in non-invasive (RT4, UROtsa) and invasive (T24, UMUC-3, and HT1197) cell lines as determined by RT-qPCR. ( B ) Immunoblot results showing IL1RA protein levels in the indicated cell lines. Both the qPCR and the Western blot yielded only one specific band at the expected size. Please note that the qPCR primers were designed to amplify all potential IL1RN isoforms. Similarly, the antibody recognizes all potential IL1RA variants. Please note that primary human tissue samples could not be tested by Western blot due to low tissue availability.

Article Snippet: The human UCB cell lines T24, UM-UC3, and RT4 were obtained from the European Collection of Authenticated Cell Cultures (ECACC).

Techniques: Expressing, Isolation, Quantitative RT-PCR, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: The Role of Interleukin-1-Receptor-Antagonist in Bladder Cancer Cell Migration and Invasion

doi: 10.3390/ijms22115875

Figure Lengend Snippet: Evaluation of migration and invasion capacity of bladder cancer cell lines by RTCA and Boyden chamber assays. Migration ( A ) and invasion ( B ) capacities of bladder cancer cell lines as determined by RTCA impedance sensing. The cell index (CI) directly correlates to the migration and invasion capacity of cells (see Materials and Methods for experimental details). The experiment was performed three times, with quadruplicates of each experiment, and similar results were obtained. Depicted are the mean CI ± SD calculated from four replicates of each experiment ( n = 3). ( C ) Modified Boyden chamber assay. Tumor cells stained with CellTracker™ Green CMFDA Dye migrate through a HUVEC-coated membrane. After 12, 24, and 48 h, T24 cells were detected in the lower well (green signal) at a much higher rate than RT4 cells. Representative immunofluorescence images of at least three independent experiments are shown.

Article Snippet: The human UCB cell lines T24, UM-UC3, and RT4 were obtained from the European Collection of Authenticated Cell Cultures (ECACC).

Techniques: Migration, Modification, Boyden Chamber Assay, Staining, Membrane, Immunofluorescence

Journal: International Journal of Molecular Sciences

Article Title: The Role of Interleukin-1-Receptor-Antagonist in Bladder Cancer Cell Migration and Invasion

doi: 10.3390/ijms22115875

Figure Lengend Snippet: Ectopic IL1RA expression impairs migration and invasion capacity of invasive T24 cell line in RTCA experiments. ( A ) Ectopic IL1RN expression in invasive T24 cells. (Left) RT-qPCR showing the ectopic IL1RN expression in infected T24-IL1RA cells (T24-pBabe-IL1RN) compared to parental T24 and mock-infected empty vector T24 (T24-pBabe-Puro) (** indicates p = 0.026). IL1RN expression in RT4 cells served as a positive control. (Right) Western blot results confirmed the overexpression of IL1RA. T24-IL1RA exhibit a strong IL1RA signal, with an intensity similar to the positive control RT4 cells. T24-negative control cells (T24 parental and T24-EV) showed no signal for IL1RA. β-ACTIN served as a loading control. ( B , C ) The graphs show migration (left) and invasion (right) capacities of T24-IL1RA (T24-pBabe IL1RN) compared to T24-EV (T24-pBabe-Puro). Wells containing serum-free media (RPMI) served as control. In the migration setting ( B ), CI values of T24-IL1RA are consistently lower than those of T24 empty vector. In the invasion setting ( C ), the difference in CI clearly manifests after 30 h. The graphs show the quantification of the cell index (mean) at the 40 h time-point. The experiment was performed two times ( n = 2) in the migration setting and three times in the invasion setting, with quadruplicates of each experiment. Depicted are the mean CI ± SD calculated from quadruplicates of one experiment. ( D ) Ectopic IL1RN expression impairs migration and invasion capacity of invasive UCB cell lines as determined by the Boyden chamber assay. Four independent repeat experiments (1st, 2nd, 3rd, and 4th) were performed to determine the impact of IL1RA on cell invasion through Matrigel after an incubation period of 48 h (left). The results were evaluated quantitatively by manually counting the cells in five randomly selected membrane areas using the image processing program ImageJ (Version 1.51). The average number of invaded cells of T24-IL1RA is significantly lower compared to T24-EV (right). ( E ) Ectopic IL1RN expression impairs the invasion capacity of the T24 cells in the porcine bladder organ culture model. Representative pictures are shown indicating invasion (black arrows) of the parental T24 (left) and T24 cells containing the empty vector (middle) into the porcine lamina propria while invasion was completely absent with the T24-IL1RA cells (right). Images were taken with the microscope Axio Imager 2 (Zeiss, Oberkochen) at 200× magnification.

Article Snippet: The human UCB cell lines T24, UM-UC3, and RT4 were obtained from the European Collection of Authenticated Cell Cultures (ECACC).

Techniques: Expressing, Migration, Quantitative RT-PCR, Infection, Plasmid Preparation, Positive Control, Western Blot, Over Expression, Negative Control, Control, Boyden Chamber Assay, Incubation, Membrane, Organ Culture, Microscopy

Journal: International Journal of Molecular Sciences

Article Title: The Role of Interleukin-1-Receptor-Antagonist in Bladder Cancer Cell Migration and Invasion

doi: 10.3390/ijms22115875

Figure Lengend Snippet: Recombinant IL1RA impairs IL1B-induced breakdown of HUVEC monolayer barrier function. ( A ) Secreted IL1RA determination. Secreted IL1RA in the supernatant (SN) of the indicated samples as determined by ELISA. The dashed line indicates the background of the ELISA signal. The results are mean values of three independent experiments. Considerable concentrations of IL1RA could be detected in the SN of RT4, UROtsa, and T24 + ILRN cells. ( B ) IL1B-induced breakdown of HUVEC monolayer barrier function is attenuated by IL1RA addition in a dose-dependent manner. The HUVEC monolayer is exposed to IL1B (1 ng/mL) or a combination of IL1B with varying concentrations of IL1RA (0.003 ng/mL, 0.03 ng/mL, 0.3 ng/mL, 3 ng/mL, 30 ng/mL, 300 ng/mL). HUVECs stimulated with serum-free media (RPMI) served as control. The CI was normalized to a time-point after cytokine addition. Depicted is the mean of normalized CI ± SD derived from four replicates in one experiment. The experiment was performed three times ( n = 3) with similar results.

Article Snippet: The human UCB cell lines T24, UM-UC3, and RT4 were obtained from the European Collection of Authenticated Cell Cultures (ECACC).

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Control, Derivative Assay

Journal: Cancer Management and Research

Article Title: Transcription factor Nrf2 induces the up-regulation of lncRNA TUG1 to promote progression and adriamycin resistance in urothelial carcinoma of the bladder

doi: 10.2147/CMAR.S200998

Figure Lengend Snippet: Clinicopathological features of patients with urothelial carcinoma of the bladder (UCB)

Article Snippet: Human UCB cell lines (EJ-1, 5637 and T24) and normal human urothelial cells (SV-HUC-1) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques:

Journal: Cancer Management and Research

Article Title: Transcription factor Nrf2 induces the up-regulation of lncRNA TUG1 to promote progression and adriamycin resistance in urothelial carcinoma of the bladder

doi: 10.2147/CMAR.S200998

Figure Lengend Snippet: Increased expression of Nrf2 and TUG1 in urothelial carcinoma of the bladder (UCB) tissues. ( A and B ) 27 pairs of UCB tissues and paired normal tissues were collected and analyzed for the expression of Nrf2 and TUG1 by RT-PCR. ( C ) Correlation analysis of Nrf2 and TUG1 expression in UCB tissues. * P <0.05.

Article Snippet: Human UCB cell lines (EJ-1, 5637 and T24) and normal human urothelial cells (SV-HUC-1) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Cancer Management and Research

Article Title: Transcription factor Nrf2 induces the up-regulation of lncRNA TUG1 to promote progression and adriamycin resistance in urothelial carcinoma of the bladder

doi: 10.2147/CMAR.S200998

Figure Lengend Snippet: Up-regulation of Nrf2 and TUG1 in ADM-resistant cells. ( A and B ) Nrf2 expression was measured in UCB cell lines (EJ-1, 5637, BIU-87 and T24) and normal human urothelial cells (SV-HUC-1) by RT-PCR and Western blot. ( C ) Western blot analysis of Nrf2 expression showed increased expression of Nrf2 in ADM-resistant cells (BIU-87/ADM and T24/ADM). ( D ) TUG1 expression was measured in UCB cell lines (EJ-1, 5637, BIU-87 and T24) and SV-HUC-1 cells by RT-PCR. ( E ) RT-PCR analysis of TUG1 expression indicated increased expression of TUG1 in BIU-87/ADM and T24/ADM cells. ** P <0.01 and *** P <0.001.

Article Snippet: Human UCB cell lines (EJ-1, 5637 and T24) and normal human urothelial cells (SV-HUC-1) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Cancer Management and Research

Article Title: Transcription factor Nrf2 induces the up-regulation of lncRNA TUG1 to promote progression and adriamycin resistance in urothelial carcinoma of the bladder

doi: 10.2147/CMAR.S200998

Figure Lengend Snippet: Nrf2 positively regulates the expression of TUG1 in UCB cells. ( A ) BIU-87 and T24 cells were transfected with pcDNA- Nrf2 , si- Nrf2 or matched controls, and the transfection efficiency was identified by Western blot. ( B ) BIU-87 and T24 cells were treated with different doses (0.1 and 0.2 μM) of ML385, and Nrf2 levels were determined by Western blot. ( C and D ) RT-PCR analysis of TUG1 expression indicated increased expression of TUG1 in BIU-87 and T24 cells transfected with pcDNA- Nrf2 . ( E and F ) RT-PCR analysis of TUG1 expression indicated decreased expression of TUG1 in BIU-87 and T24 cells transfected with si- Nrf2 . (G and H) RT-PCR analysis of TUG1 expression showed down-regulation of TUG1 in BIU-87 and T24 cells treated with ML385. * P <0.05, ** P <0.01 and *** P <0.001.

Article Snippet: Human UCB cell lines (EJ-1, 5637 and T24) and normal human urothelial cells (SV-HUC-1) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, Transfection, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: Cancer Management and Research

Article Title: Transcription factor Nrf2 induces the up-regulation of lncRNA TUG1 to promote progression and adriamycin resistance in urothelial carcinoma of the bladder

doi: 10.2147/CMAR.S200998

Figure Lengend Snippet: Knockdown of either Nrf2 or TUG1 inhibits the progression of UCB in vitro. BIU-87 and T24 cells were transfected with si- Nrf2 , si- TUG1 or si-NC. ( A ) Cell viability was analyzed at the indicated time-points (0, 1, 2, 3 days after transfection) by CCK-8 assay. ( B and C ) Ki-67 expression was measured 48 h post-transfection by Western blot. ( D and E ) The invasion ability of BIU-87 and T24 cells was determined 48 h post-transfection by transwell invasion assay. ( F and G ) The protein levels of MMP-2 and MMP-9 were examined by Western blot. ( H and I ) Cell apoptosis was analyzed 48 h post-transfection by flow cytometry. ( J and K ) Cleaved caspase-3 levels were evaluated in BIU-87 and T24 cells transfected with si- Nrf2 or si- TUG1 by Western blot.* P <0.05, ** P <0.01 and *** P <0.001.

Article Snippet: Human UCB cell lines (EJ-1, 5637 and T24) and normal human urothelial cells (SV-HUC-1) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Knockdown, In Vitro, Transfection, CCK-8 Assay, Expressing, Western Blot, Transwell Invasion Assay, Flow Cytometry

Journal: Cancer Management and Research

Article Title: Transcription factor Nrf2 induces the up-regulation of lncRNA TUG1 to promote progression and adriamycin resistance in urothelial carcinoma of the bladder

doi: 10.2147/CMAR.S200998

Figure Lengend Snippet: Knockdown of either Nrf2 or TUG1 enhances the chemosensitivity of ADM-resistant UCB cells to ADM. ( A ) RT-PCR analysis of MDR1 mRNA expression indicated up-regulation of MDR1 in BIU-87/ADM and T24/ADM cells. ( B ) Western blot analysis of p-gp expression showed elevated expression of p-gp in BIU-87/ADM and T24/ADM cells. ( C ) BIU-87/ADM and T24/ADM cells were transfected with si- Nrf2 , si- TUG1 or si-NC, followed by exposure to indicated doses (0, 5, 10, 20 and 40 μg/ml) of ADM. Cell viability was determined using CCK-8 assay. ( D and E ) BIU-87/ADM and T24/ADM cells were transfected with si- Nrf2 , si- TUG1 or si-NC. At 48 h post transfection, the expression of p-pg was evaluated by Western blot. * P <0.05, ** P <0.01 and *** P <0.001.

Article Snippet: Human UCB cell lines (EJ-1, 5637 and T24) and normal human urothelial cells (SV-HUC-1) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Knockdown, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Transfection, CCK-8 Assay

Journal: Cancer Management and Research

Article Title: Transcription factor Nrf2 induces the up-regulation of lncRNA TUG1 to promote progression and adriamycin resistance in urothelial carcinoma of the bladder

doi: 10.2147/CMAR.S200998

Figure Lengend Snippet: Knockdown of either Nrf2 or TUG1 enhances the sensitivity of UCB cells to ADM in vivo. T24/ADM cells stably expressing sh- Nrf2 , sh- TUG1 or sh-NC were subcutaneously injected into nude mice. Subsequently, mice were intraperitoneally injected with ADM or saline. ( A ) Tumor growth curves of T24/ADM cells transfected with sh- Nrf2 , sh- TUG1 or sh-NC and treated with ADM or saline i n vivo . ( B ) The mean tumor weight of the six groups. ( C ) Western blot analysis was performed to examine expression of Nrf2 protein in xenografted tumors. ( D and E ) RT-PCR analysis of TUG1 and MDR1 mRNA expression in xenografted tumors. ( F ) Western blot analysis of p-gp protein expression in xenografted tumors. * P <0.05, ** P <0.01 and *** P <0.001.

Article Snippet: Human UCB cell lines (EJ-1, 5637 and T24) and normal human urothelial cells (SV-HUC-1) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Knockdown, In Vivo, Stable Transfection, Expressing, Injection, Saline, Transfection, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: Cancer Management and Research

Article Title:

Transcription factor Nrf2 induces the up-regulation of lncRNA TUG1 to promote progression and adriamycin resistance in urothelial carcinoma of the bladder

doi: 10.2147/cmar.s200998

Figure Lengend Snippet: Figure 1 Increased expression of Nrf2 and TUG1 in urothelial carcinoma of the bladder (UCB) tissues. (A and B) 27 pairs of UCB tissues and paired normal tissues were collected and analyzed for the expression of Nrf2 and TUG1 by RT-PCR. (C) Correlation analysis of Nrf2 and TUG1 expression in UCB tissues. *P<0.05.

Article Snippet: Cell culture Human UCB cell lines (EJ-1, 5637 and T24) and normal human urothelial cells (SV-HUC-1) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Cancer Management and Research

Article Title:

Transcription factor Nrf2 induces the up-regulation of lncRNA TUG1 to promote progression and adriamycin resistance in urothelial carcinoma of the bladder

doi: 10.2147/cmar.s200998

Figure Lengend Snippet: Figure 2 Up-regulation of Nrf2 and TUG1 in ADM-resistant cells. (A and B) Nrf2 expression was measured in UCB cell lines (EJ-1, 5637, BIU-87 and T24) and normal human urothelial cells (SV-HUC-1) by RT-PCR and Western blot. (C) Western blot analysis of Nrf2 expression showed increased expression of Nrf2 in ADM-resistant cells (BIU-87/ADM and T24/ADM). (D) TUG1 expression was measured in UCB cell lines (EJ-1, 5637, BIU-87 and T24) and SV-HUC-1 cells by RT-PCR. (E) RT-PCR analysis of TUG1 expression indicated increased expression of TUG1 in BIU-87/ADM and T24/ADM cells. **P<0.01 and ***P<0.001.

Article Snippet: Cell culture Human UCB cell lines (EJ-1, 5637 and T24) and normal human urothelial cells (SV-HUC-1) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Cancer Management and Research

Article Title:

Transcription factor Nrf2 induces the up-regulation of lncRNA TUG1 to promote progression and adriamycin resistance in urothelial carcinoma of the bladder

doi: 10.2147/cmar.s200998

Figure Lengend Snippet: Figure 3 Nrf2 positively regulates the expression of TUG1 in UCB cells. (A) BIU-87 and T24 cells were transfected with pcDNA-Nrf2, si-Nrf2 or matched controls, and the transfection efficiency was identified by Western blot. (B) BIU-87 and T24 cells were treated with different doses (0.1 and 0.2 μM) of ML385, and Nrf2 levels were determined by Western blot. (C and D) RT-PCR analysis of TUG1 expression indicated increased expression of TUG1 in BIU-87 and T24 cells transfected with pcDNA-Nrf2. (E and F) RT- PCR analysis of TUG1 expression indicated decreased expression of TUG1 in BIU-87 and T24 cells transfected with si-Nrf2. (G and H) RT-PCR analysis of TUG1 expression showed down-regulation of TUG1 in BIU-87 and T24 cells treated with ML385. *P<0.05, **P<0.01 and ***P<0.001.

Article Snippet: Cell culture Human UCB cell lines (EJ-1, 5637 and T24) and normal human urothelial cells (SV-HUC-1) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, Transfection, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: Cancer Management and Research

Article Title:

Transcription factor Nrf2 induces the up-regulation of lncRNA TUG1 to promote progression and adriamycin resistance in urothelial carcinoma of the bladder

doi: 10.2147/cmar.s200998

Figure Lengend Snippet: Figure 4 Knockdown of either Nrf2 or TUG1 inhibits the progression of UCB in vitro. BIU-87 and T24 cells were transfected with si-Nrf2, si-TUG1 or si-NC. (A) Cell viability was analyzed at the indicated time-points (0, 1, 2, 3 days after transfection) by CCK-8 assay. (B and C) Ki-67 expression was measured 48 h post-transfection by Western blot. (D and E) The invasion ability of BIU-87 and T24 cells was determined 48 h post-transfection by transwell invasion assay. (F and G) The protein levels of MMP-2 and MMP-9 were examined by Western blot. (H and I) Cell apoptosis was analyzed 48 h post-transfection by flow cytometry. (J and K) Cleaved caspase-3 levels were evaluated in BIU-87 and T24 cells transfected with si-Nrf2 or si-TUG1 by Western blot.*P<0.05, **P<0.01 and ***P<0.001.

Article Snippet: Cell culture Human UCB cell lines (EJ-1, 5637 and T24) and normal human urothelial cells (SV-HUC-1) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Knockdown, In Vitro, Transfection, CCK-8 Assay, Expressing, Western Blot, Transwell Invasion Assay, Cytometry

Journal: Cancer Management and Research

Article Title:

Transcription factor Nrf2 induces the up-regulation of lncRNA TUG1 to promote progression and adriamycin resistance in urothelial carcinoma of the bladder

doi: 10.2147/cmar.s200998

Figure Lengend Snippet: Figure 5 Knockdown of either Nrf2 or TUG1 enhances the chemosensitivity of ADM-resistant UCB cells to ADM. (A) RT-PCR analysis of MDR1 mRNA expression indicated up- regulation of MDR1 in BIU-87/ADM and T24/ADM cells. (B) Western blot analysis of p-gp expression showed elevated expression of p-gp in BIU-87/ADM and T24/ADM cells. (C) BIU- 87/ADM and T24/ADM cells were transfected with si-Nrf2, si-TUG1 or si-NC, followed by exposure to indicated doses (0, 5, 10, 20 and 40 μg/ml) of ADM. Cell viability was determined using CCK-8 assay. (D and E) BIU-87/ADM and T24/ADM cells were transfected with si-Nrf2, si-TUG1 or si-NC. At 48 h post transfection, the expression of p-pg was evaluated by Western blot. *P<0.05, **P<0.01 and ***P<0.001.

Article Snippet: Cell culture Human UCB cell lines (EJ-1, 5637 and T24) and normal human urothelial cells (SV-HUC-1) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Knockdown, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Transfection, CCK-8 Assay

Journal: Cancer Management and Research

Article Title:

Transcription factor Nrf2 induces the up-regulation of lncRNA TUG1 to promote progression and adriamycin resistance in urothelial carcinoma of the bladder

doi: 10.2147/cmar.s200998

Figure Lengend Snippet: Figure 6 Knockdown of either Nrf2 or TUG1 enhances the sensitivity of UCB cells to ADM in vivo. T24/ADM cells stably expressing sh-Nrf2, sh-TUG1 or sh-NC were subcutaneously injected into nude mice. Subsequently, mice were intraperitoneally injected with ADM or saline. (A) Tumor growth curves of T24/ADM cells transfected with sh-Nrf2, sh-TUG1 or sh-NC and treated with ADM or saline in vivo. (B) The mean tumor weight of the six groups. (C) Western blot analysis was performed to examine expression of Nrf2 protein in xenografted tumors. (D and E) RT-PCR analysis of TUG1 and MDR1 mRNA expression in xenografted tumors. (F) Western blot analysis of p- gp protein expression in xenografted tumors. *P<0.05, **P<0.01 and ***P<0.001.

Article Snippet: Cell culture Human UCB cell lines (EJ-1, 5637 and T24) and normal human urothelial cells (SV-HUC-1) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Knockdown, In Vivo, Stable Transfection, Expressing, Injection, Saline, Transfection, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: Clinical Cancer Research

Article Title: PRMT5 Circular RNA Promotes Metastasis of Urothelial Carcinoma of the Bladder through Sponging miR-30c to Induce Epithelial–Mesenchymal Transition

doi: 10.1158/1078-0432.ccr-18-1270

Figure Lengend Snippet: Figure 2. Silencing of circPRMT5 RNA inhibited UCB cells' invasion. A, Schematic illustration showed two targeted shRNAs. Sh-circPRMT5 targets the back-splice junction of circPRMT5 (left). qRT-PCR for circPRMT5 and PRMT5 mRNA in UCB cells treated with two shRNAs as described above. Data are the mean SD of three experiments. B, qRT-PCR for circPRMT5 and PRMT5 mRNA in UCB cells transfected with control vector or circPRMT5 overexpression plasmid. Data are the mean SD of three experiments. C, Wound-healing assay showed that knockdown of circPRMT5 inhibited the migration ability of the indicated UCB cells. D, Wound-healing assay analyses showed that overexpression of circPRMT5 promoted the migration ability of the indicated UCB cells. E, Transwell assay showed that knockdown of circPRMT5 inhibited the invasion ability of the indicated UCB cells. F, Transwell assay showed that overexpression of circPRMT5 promoted the invasion ability of the indicated UCB cells. G and H, Stably transfected T24 cells with circPRMT5-shRNA (G) or overexpression of circPRMT5 (H) with their respective control lentivirus were injected into the vein of BALB/c nude mice for 6 weeks. Representative images of lungs (left), metastatic nodules (indicated by arrows), and hematoxylin and eosin (H&E) staining of lung metastatic lesions (middle, original magnification, 100; scale bar, 100 mm) are shown. The number of metastatic nodules formed in the lungs of BALB/c nude mice is summarized for each group tested (n ¼ 5; bottom right; , P < 0.01; , P < 0.001; , P < 0.0001).

Article Snippet: Cell lines and cell culture Four UCB cell lines (i.e., T24, TCC-SUP, 5637, and UM-UC-3) and a nontumorous bladder cell line, SV-HUC-1, were purchased from ATCC in 2013.

Techniques: Quantitative RT-PCR, Transfection, Control, Plasmid Preparation, Over Expression, Wound Healing Assay, Knockdown, Migration, Transwell Assay, Stable Transfection, shRNA, Injection, Staining

Journal: Clinical Cancer Research

Article Title: PRMT5 Circular RNA Promotes Metastasis of Urothelial Carcinoma of the Bladder through Sponging miR-30c to Induce Epithelial–Mesenchymal Transition

doi: 10.1158/1078-0432.ccr-18-1270

Figure Lengend Snippet: Figure 4. CircPRMT5 served as a sponge for miR-30c in UCB cells. A, Ago2 RIP assay for the amount of circPRM5 in UCB cells expressing Flag-AGO2 or Flag-GFP. Data are the mean SD of three experiments. , P < 0.0001, Student t test. B, The entire circPRMT5 sequence (red) was cloned into the downstream region of the luciferase gene, denoting LUC-circPRMT5. Luciferase reporter assay for the luciferase activity of LUC-circPRMT5 in T24 cells cotransfected with shRNA against circPRMT5 or control shRNA. Data are the mean SD of three experiments. , P < 0.001, Student t test. C, Luciferase reporter assay for the luciferase activity of LUC-circPRMT5 in T24 cells transfected with a library of 83 miRNA mimics to identify miRNAs that were able to bind to the circPRMT5 sequence. miRNAs that inhibited luciferase activity by 50% are indicated by reddots.D, A schematic drawing showing the putative binding sitesofthe miRNAs associatedwith circPRMT5. E, Luciferase reporter assay forthe luciferase activity of LUC-circPRMT5 or LUC-circPRMT5 mutant in T24 cells cotransfected with miRNA mimics. Data are the mean SD of three experiments. , P < 0.01; , P < 0.001, Student t test. F, RNA pull-downassay forthe amount of circPRMT5 and miR-30c with eithercircPRMT5 orcontrolprobe in UCB cells. RNAs that were pulled down were compared with a 10-fold dilution of input RNA and defined as percentage of input (%input). Data are normalized to that with control probe, showing the mean SD of three experiments. , P < 0.01; , P < 0.001, Student t test. G, RNA pull-down assay for the amount of circPRMT5 and miR-30c with circPRMT5probeinUCBcells expressingeithercontrolorcircPRMT5shRNA.DataarenormalizedtothatwithcontrolshRNA,showingthemean SDofthreeexperiments. , P < 0.01; , P < 0.001, Student t test. H, IF images represented RNA colocalization of circPRMT5 and miR-30c in control cells or circPRMT5-overexpressing cells.

Article Snippet: Cell lines and cell culture Four UCB cell lines (i.e., T24, TCC-SUP, 5637, and UM-UC-3) and a nontumorous bladder cell line, SV-HUC-1, were purchased from ATCC in 2013.

Techniques: Expressing, Sequencing, Clone Assay, Luciferase, Reporter Assay, Activity Assay, shRNA, Control, Transfection, Binding Assay, Mutagenesis, Pull Down Assay

Journal: Oncogene

Article Title: Overexpression of maelstrom promotes bladder urothelial carcinoma cell aggressiveness by epigenetically downregulating MTSS1 through DNMT3B.

doi: 10.1038/onc.2016.165

Figure Lengend Snippet: Figure 1. Expression and amplification of MAEL in UCB cell lines and bladder tissues and its prognostic significance in UCB patients. (a) The levels of MAEL protein examined by western blotting in seven human UCB cell lines (that is, Biu-87, UMUC3, EJ, 5637, T24, RT4 and J82) and one normal uroepithelial cell line, SV-HUC-1 (left) and in 10 UCB tissues and paired normal bladder tissues. N, normal tissue; T, carcinoma tissue (right). (b) Representative IHC images show normal expression of MAEL in adjacent normal bladder tissue and overexpression of MAEL in UCB tissues (case 1 and case 2); scale bar, 100 μm. Amplification of MAEL gene was examined by FISH in UCB tissue case 2; scale bar, 20 μm. (c) Kaplan–Meier analysis indicating the correlation of MAEL overexpression with poorer overall survival and disease-free survival rates of 184 patients with UCB (log-rank test).

Article Snippet: Human UCB cell line T24, 5637, UMUC3, J82 and RT4, and human normal uroepithelial cell line SV-HUC-1 was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, Western Blot, Over Expression

Journal: Oncogene

Article Title: Overexpression of maelstrom promotes bladder urothelial carcinoma cell aggressiveness by epigenetically downregulating MTSS1 through DNMT3B.

doi: 10.1038/onc.2016.165

Figure Lengend Snippet: Figure 2. MAEL has strong oncogenic function in UCB cells. (a) Western blotting reveals that MAEL was efficiently knocked down by the treatment of MAEL-shRNA-1 or MAEL-shRNA-2 in T24 and UMUC3 UCB cells. (b) Rate of cell growth between shMAEL and shControl UCB cells by CCK-8 kit. *Po0.05, **Po0.01 by Student's t-test. (c) Representative images of decreased foci formation in monolayer culture induced by MAEL silenced in UCB cells. Data are the means ± s.d. of three independent experiments. **Po0.01 by Student's t-test. (d) The levels of MAEL were substantially increased in 5637-MAEL cells compared with that in control 5637-vector cells by western blotting (left). Overexpression of MAEL promoted 5637 cells growth rate (right). **Po0.01 by Student's t-test. (e) Representative images of increased foci formation in monolayer culture induced by MAEL overexpression in 5637 cells. Data are the mean ± s.d. of three independent experiments. **Po0.01 by Student's t-test. (f) Images of the xenograft tumors formed in nude mice injected with UMUC3-shControl, UMUC3-MAEL-shRNA-1 cells (upper) and 5637-vector, 5637-MAEL cells (down). Weights of xenograft tumors are the mean ± s.d. **Po0.01 by Student's t-test.

Article Snippet: Human UCB cell line T24, 5637, UMUC3, J82 and RT4, and human normal uroepithelial cell line SV-HUC-1 was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Western Blot, shRNA, CCK-8 Assay, Control, Plasmid Preparation, Over Expression, Injection

Journal: Oncogene

Article Title: Overexpression of maelstrom promotes bladder urothelial carcinoma cell aggressiveness by epigenetically downregulating MTSS1 through DNMT3B.

doi: 10.1038/onc.2016.165

Figure Lengend Snippet: Figure 4. MTSS1 is responsible for MAEL-induced UCB cell invasiveness and EMT. (a) The seven genes, EWSR1, MTSS1, MDM2, MMP2, Flt4, CCL7 and FGFR4, were examined 42-fold mRNA differential expression in T24-MAEL-shRNA-1 cells compared with that in T24-shControl cells by using a Human Tumor Metastasis RT2 Profiler PCR Array. (b) Silence of MAEL by shRNA-1 in T24 cells substantially upregulated MTSS1 expression and downregulated Flt4 expression as examined by western blotting. (c) Overexpression of MAEL and low-level expression of MTSS1 was examined by IHC in a UCB case; scale bar, 100 μm. (d) Western blot shows that MTSS1 was efficiently knocked down by the treatment of siMTSS1 in T24-MAEL-shRNA-1 cells. (e) Wound-healing and Transwell assays show that knockdown of MTSS1 substantially increased the migration (upper) and invasion (down) abilities of T24-MAEL-shRNA-1 cells. Data are the means ± s.d. of three independent experiments; scale bar, 100 μm. *Po0.05, **Po0.01 by Student's t-test. (f) Western blotting shows that after silence of MTSS1 by specific siRNA in T24-MAEL-shRNA-1 cells, the levels of E-cadherin and β-catenin decreased, whereas the levels of fibronectin and vimentin increased (left). IF staining demonstrates a downregulated expression of E-cadherin and an upregulated expression of vimentin in T24-MAEL-shRNA-1 cells, after knockdown of MTSS1 (right).

Article Snippet: Human UCB cell line T24, 5637, UMUC3, J82 and RT4, and human normal uroepithelial cell line SV-HUC-1 was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Quantitative Proteomics, shRNA, Expressing, Western Blot, Over Expression, Knockdown, Migration, Staining